Infections due to Neisseria meningitidis remain a worldwide health problem, especially for children and young adults. N. meningitidis is an exclusive human pathogen which has evolved strategies to infect human mucosal surfaces such as the nasopharynx. Lipopolysaccharide, also called lipooligosaccharide (LOS), is known to be an important virulence factor for meningococcal infections. However, the genetic basis for expression of meningococcal LOS, and how LOS is involved in the events resulting in meningococcal infection of the human nasopharynx, are poorly understood. Recently, we found that the 16.4 kb conjugative transposon Tn916 can be introduced into N. meningitidis by transformation of the suicide vector pAM120. Tn916 appears to transpose as a single copy to many different sites in the chromosome of recipient meningococci and is stably integrated. The Specific Aims of this project are to: 1) identify, using Tn916 mutagenesis, genetic determinants that contribute to the structure of meningococcal LOS and 2) use genetically defined mutations in LOS structure to elucidate the role of LOS in meningococcal attachment, invasion and cytotoxicity of human cells. To accomplish these aims, our library of Tn916-containing meningococcal mutants will be expanded to achieve a maximum number of different mutations. Monoclonal antibodies and SDS-PAGE will be used to identify Tn916 mutants which are altered in structure of LOS. Linkage of the Tn916 insertion and the LOS phenotype will be demonstrated by transformation and Southern blots. The LOS mutants will be characterized by generation time, outer membrane protein profile and piliation. The site(s) of Tn916 in the genome of the LOS mutants will be analyzed by Southern blots and PCR. The nucleotide sequence of meningococcal DNA adjacent to the transposon will be obtained from cloned inserts or SSP-PCR. The gene in the parental strain will be identified and the specificity of the determinant for altering LOS structure confirmed by insertional inactivation of the gene. The LOS genetic determinant will be characterized by analysis of nucleotide sequence, gene product and distribution. Using in vitro attachment assays and human nasopharyngeal organ cultures, the effect of altered LOS structure on meningococcal attachment, invasion and cytotoxicity of human cells will then be studied. Understanding the molecular basis for meningococcal virulence is needed to develop new strategies to better control meningococcal infections. These studies should also provide insight into the genetic basis of LOS structure, help unravel how meningococci affect eucaryotic cellular pathways and continue development of a biologically relevant in vitro model for studies of microbial pathogenesis.